P63 and Ki-67 Expression in Dentigerous Cyst and Ameloblastomas

Statement of the Problem P63 gene is a member of TP53 and its homologous gene family. Its expression was observed in some odontogenic lesions, more expression in aggressive lesions. Purpose This study aimed to investigate the possible diagnostic impact of P63 protein on dentigerous cysts and various types of ameloblastoma. Its expression with Ki-67 proliferation marker was also compared. Materials and Method This cross-sectional retrospective study was enrolled on 25 cases of dentigerous cyst including 21 unicystic ameloblastomas and 17 conventional ameloblastomas. The expression of P63 and Ki-67 was assessed by immunohistochemical (IHC) examinations. Data were analyzed by employing Mann-Whitney and correlation coefficient tests. Results P63 expression was significantly higher in ameloblastoma than unicystic ameloblastoma and dentigerous cysts. There was no significant difference between unicystic ameloblastoma and dentigerous cyst in P63 expression. A 90% cut-off point was obtained for basal layer which gave 88% sensitivity and 78% specificity to distinguish more invasive lesions from others. There was not any correlation between P63 and Ki-67 immunostaining in the three study groups. Conclusion More aggressiveness and more invasiveness of odontogenic lesions depicted higher rate and also more intensive expression of P63. Moreover, the expression of P63 protein had not any correlation with Ki-67 protein in dentigerous cysts and ameloblastomas.


Introduction
Odontogenic cysts and tumours arise from the odontogenic epithelium of tooth germ. Ameloblastoma is the most common odontogenic tumour with clinical significance. This neoplasm appears in three forms: conventional, unicystic and peripheral. These lesions present different clinical and histopathological features which need different managements. Conventional type is a locally invasive benign tumour with high recurrence rate. [1][2] Unicystic ameloblastoma mimics the dentigerous cyst in clinical, radiographical, and even histo-pathological features. Unicystic ameloblastoma may arise from a dentigerous cyst, but these odontogenic lesions have different clinical behaviours and treatment managements. Therefore, accurate diagnosis and identifying the processes which explain the tumour growth and invasion are the matter of concern. TP63 (p63) is a homologue of TP53 gene and is located at the 3q27-29 locus. P63 has two promoters and produces two types of protein: TAP63 that contains acidic N-terminal trans-activation domain and ∆NP63 that lacks this domain. [3][4] Expression of P63 protein is discovered in skin, oesophagus, oral mucosa, prostate, breast, lung, salivary glands, and odontogenic epithelium of tooth germ as well as dental follicle of impacted teeth. [5][6][7] Studies showed that P63 is an essential protein for epithelial stratification [8] and various isoforms of P63 have different roles. ∆NP63 proteins contribute to cell proliferation; while, TAP63 isoforms induce cell differentiation. [9] There are some researches on expression of P63 in odontogenic cysts and tumours. [10][11][12][13][14] They reported that more aggressive tumours such as keratocystic odontogenic tumour (KCOT) and ameloblastoma have more expression of P63. [10][11][12] However, most of these studies demonstrated semi-quantitative data and did not present any cut-off point to help diagnosis. Ameloblastomas, unicystic and solid types, may arise from a dentigerous cyst and may show transitional changes from a non-aggressive cyst to a locally invasive tumour. Therefore, we analyzed the comparative expression of P63 in these lesions to evaluate this protein as a marker in early diagnosis which consequently helps selecting accurate management. Ki-67 is the most frequently applied proliferation marker for evaluating proliferative activity and biologic behaviour of many pathologic lesions, including odontogenic cysts and tumours. [15][16] Regarding the role of P63 protein in epithelial cell proliferation, we also evaluated the correlation of Ki-67 and P63 in those odontogenic lesions.

Statistical analysis
Data was analysed by using SPSS software (version 11).
T-test, Mann-Witney, and correlation tests were used as appropriated. P-value<0.05 was considered as significant. A receiver operating characteristic (ROC) curve was obtained to distinguish more invasive lesions (mural and solid ameloblastoma) from other non-aggressive lesions.

Results
The  Tables 1 and 2. There were 5 cases with mild to moderate inflammation that showed a slightly lower P63-expression, but it was not significantly different with non-inflamed cysts (p> 0.05).   There was no significant difference in overall P63 expression between dentigerous cyst and unicystic ameloblastoma; however, T-test analysis showed a statistically significant difference between P63 expression in dentigerous cyst and luminal unicystic ameloblastoma in suprabasal (p= 0.02), but not in basal layers (p= 0.5).
Also, peripheral cells of ameloblastic nests in solid and mural ameloblastomas revealed a significant difference in labelling P63 (p= 0.01), but not in central cells.
Overall P63 expression was significantly higher in ameloblastoma than unicystic ameloblastoma and dentigerous cyst according to t-test (p= 0.04 and 0.002, respectively). The intensity of staining was evaluated by using Chi-square test (Table 2) and the results showed that intensity was significantly different between the three groups (p= 0.001); however, there was no difference between various tumour subtypes.
Solid ameloblastoma had significantly higher LI than unicystic ameloblastoma and dentigerous cyst. The difference in Ki-67 LI between dentigerous cyst and unicystic ameloblastomas was not statistically significant.

The correlation test revealed no correlation between P63
and Ki-67 expression in the study groups.   (Figure 3).

Discussion
In this study, the expression of P63 protein was evaluat- In the present study, the immune reaction of P63 was slightly lower than that in inflamed cysts, though not statistically significant. Gonçalves et al. found this finding in the cases of severe inflamed radicular cyst.
In a similar way, the infiltrating nests in mural ameloblastoma and solid type showed severe staining in most of the peripheral and many central cells. Mural and solid ameloblastomas are locally infiltrative neoplasms which need a more invasive surgical treatment than dentigerous cyst and luminal unicystic ameloblastoma.
Moreover, we analyzed this marker as a diagnostic aid to distinguish aggressive from non-aggressive odontogenic lesions which have clinicopathological similarities. According to the results, 90% or more staining in the basal layer supported mural and solid ameloblastomas, which should be considered for more extensive surgical management and longer follow-up than nonaggressive cystic lesions. Our results showed that evaluation of basal layer was more accurate than suprabasal layers for differentiation of these lesions. These results may be useful in small biopsied specimens in which the final diagnosis is not simple. Our results support the hy-pothesis which construed that P63 protein may contribute to the tumour genesis of odontogenic structures. [13] In the present study, less differentiated cells that